A) Field of the Invention
This invention relates to the separation of cellular constituents and more particularly relates to the isolation of certain nucleic acids and peptides from other cellular materials. The invention, for example, concerns the isolation of plasmids, RNA's, mitochondrial DNA's, viral DNA's, chloroplast DNA's, other episomal DNA's and certain proteins.
B) History of the Prior Art
Several different procedures have been developed for the isolation of bacterial plasmids and other nucleic acids and proteins from cellular materials. For example, plasmids can be purified from cleared lysates of bacterial cells by centrifuging in density gradients, e.g., cesium chloride and ethidium bromide. Alternatively, nucleic acids such as plasmids can be isolated from bacterial lysates by selective precipitation from high saltsodium dodecyl sulfate systems, by differential alkaline denaturation, by selective extraction in phenol or by hydroxyapatite chromatography. For various references relating to the isolation of nucleic acids from other cellular materials see "Supercoiled Circular DNA-Protein Complex In Escherichia Coli: Purification And Induced Conversion To An Open Circular DNA Form" by D. B. Clewell and D. R. Helinski, Proceedings of The National Academy of Science USA, Volume 62, Pages 1159 to 1166; "Selective Extraction of Polyoma DNA from Infected Mouse Cell Cultures" by B. Hirt (1967) Journal of Molecular Biology, Volume 26, Pages 365 to 369; "Isolation of Covalently Closed Circular DNA of High Molecular Weight from Bacteria" by T. C. Currier and E. W. Nester (1976), Analytical Biochemistry, Volume 76, Pages 431 to 441; "A New Method for the Purification and Identification of Covalently Closed Circular DNA Molecules" by M. Zasloff, G. Ginder and G. Felsenfeld (1978), Nucleic Acids Research, Volume 5, Pages 1139 to 1152; "Rapid Purification of Plasmid DNAs by Hydroxyapatite Chromatography" by A. Colman et al (1978), European Journal of Biochemistry, Volume 91, Pages 303 to 310; and "A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA" by H. C. Birnboim et al (1979) Nucleic Acids Research, Volume 7, 1513 to 1523.
The procedures for isolating plasmids and other nucleic acids in protein described in the above references are quite complicated and expensive. Furthermore, in most cases, the procedures for such isolations are extremely time consuming.
The need for isolation of plasmids and other nucleic acids has become critical due to the extremely rapid growth of microbiological analysis and genetic engineering. Isolated plasmids are especially in demand since plasmids provide one of the simpliest paths for introducing biological functions into living organisms. In particular, plasmids are cleaved with restriction enzymes followed by the introduction of a nucleic acid grouping at the cleaved site which carries the code for a particular biological function. The plasmid ring is then again closed and the plasmid is introduced into a living organism which then carries out the coded biological function introduced into the plasmid.